The goal of this research is to improve selective chemotherapy for head and neck (HN) squamous cell carcinoma (SCC) by targeting the folate- dependent enzyme thymidylate synthase (TS). Current antifolate therapy (methotrexate; MTX) is used in HNSCC, but resistance often occurs to chemotherapy regimens in which this dihydrofolate reductase (DHFR) inhibitor is employed thus limiting both response rate and long-term survivors. However, since folate metabolism is vulnerable in HNSCC, antifolates unrelated to MTX may be therapeutically superior. The folate- dependent enzyme TS, because of its role as the only de novo source of thymidine nucleotides for DNA synthesis, is a key target for new antifolates. We propose to explore the cellular/biochemical pharmacology of three new TS-directed inhibitors in in vitro human HNSCC models. These data will be used to optimally design therapeutic studies in mice proposed in Project 2 of this Interactive RO1 (Dr. Y. Rustum) and to define crucial determinants of response to these drugs that should be monitored in vivo (Project 2). This goal will be addressed through these Specific Aims: 1. Characterize human HNSCC cell lines to develop model systems for studying TS inhibitors. FaDu, from a human pharynx SCC, and A253, from a human epidermoid carcinoma of the neck, cell lines will be used since they represent intrinsic sensitivity (FaDu) and resistance (A253) to MTX in brief exposures, such as occur clinically. Another factor in their choice is that A253 and FaDu produce well-differentiated and less differentiated squamous cell carcinomas, respectively, in nude mice and thus can be used in in vivo studies (Project 2). Growth characteristics, cloning efficiency, and effects of supplements will be assessed during culture as monolayers and as multicell spheroids (MS). 2. Define the cellular and biochemical pharmacology in human HNSCC models and normal murine intestine of D1694, AG331, and 1843U89 (three folate- related TS inhibitors) as single agents. Concentration/schedule dependence of monolayer HNSCC cell growth inhibition by the drugs as single agents will be fully defined; clonogenic survival will be measured as appropriate. The mechanism of action of the drugs will be studied using isolated enzymes (TS, folylpolyglutamate synthetase, and DHFR), metabolite protection, and measurement of whole cell transport/metabolism and folate pools to identify crucial determinants of response. Other parameters would be investigated as dictated by results. Analogous studies in HNSCC MS, would be undertaken. Results from monolayer and MS studies will be correlated with in vivo results (Project 2). Specific comparative studies using normal mouse intestinal epithelium will be used to explore selective toxicity of these drugs. Useful combinations of TS inhibitors and selected agents would be studied in a renewal. 3. Select sublines of the HNSCC models with acquired resistance to each TS inhibitor and characterize the resistance phenotype. Sublines of HNSCC models resistant to the drugs will be selected in monolayers using both continuous and intermittent drug exposure, since the exposure schedule may influence resistance frequency and/or resistance phenotype. Biochemical/cellular study of resistant sublines will be performed as for parental cells. Studies of resistance in the HNSCC MS model would be initiated following a similar protocol. Resistance in both in vitro models will be correlated with resistance in vivo (Project 2).